Round-bottom Plates Research Articles

Abstract 4223: The dual immunestimulatory and immunesuppressive role of MRC5 fibroblast cells in 3D coculture with T cells and tumor cells

Abstract Introduction: Cancer-associated fibroblasts (CAFs) alter anti-tumor immune responses and modulate resistance to immune checkpoint inhibitors. The mechanisms of crosstalk between tumor cells, CAFs, and T cells remain not fully understood, and studying these multicellular interactions requires adoption of novel tools in preclinical research. Here, we established a spheroid co-culture model comprising of tumor cell lines, a fibroblast cell line, and primary human T cells, for studying the immunomodulatory effects of fibroblasts on T cell responses in vitro, in the context of immunotherapies. Methods: For setting up the 3D co-culture, A375 or OV-90 tumor cells were seeded alone or with MRC-5 fibroblasts in 96-well ultra-low attachment round bottom plates in complete growth medium supplemented with 1% Geltrex matrix. The cells were centrifuged, and spheroids were allowed to form for 24 hours. After spheroid initiation, activated T cells and treatments with anti-PD1 antibody or an immune stimulatory drug were added. Spheroid co-cultures were live imaged by Incucyte SX5 for 72 hours taking images every 4 hours, after which treatment responses were evaluated by flow cytometry and measuring IFN-γ accumulation with ELISA. Results: To mimic fibroblast and T cell interactions in tumor microenvironment we built and optimized an in vitro 3D co-culture assay using various tumor cell lines. With this assay we found that depending on the tumor cell interaction, fibroblasts MRC-5 cell line have either immune-suppressive or immune-promoting properties. IFN-γ secretion of T cells in the assay indicated that MRC-5 cells suppressed T cells when co-cultured with A375 cells, while with OV-90 cells T cells activation was enhanced. In addition to the IFN- γ secretion, T cells in MRC-5 OV-90 interaction significantly upregulated 4-1BB expression on both CD4 & CD8 T cells. Interestingly we noticed that MRC-5 fibroblasts could modulate T cell responses to anti-PD-1 treatment in the co-culture depending on the tumor cell used. Conclusion: Here, we established a spheroid co-culture model, which will allow studies of the immunomodulatory crosstalk occurring in tumor microenvironment. Our model revealed a bifunctional role of MRC-5 fibroblasts in altering T cell phenotype. This finding is in line with literature highlighting the ability of fibroblasts to act as either tumor promoters or tumor suppressors depending on tumor microenvironment. We believe that this model, would aid in elucidating mechanisms of interaction between CAFs and T cells, potentially leading to discoveries of novel targets for immuno-oncology drug development. Additional studies, such as gene expression analysis using single cell RNA sequencing, are warranted to further validated the model and to better understand the kinetics of these interaction occurring in the co-culture with various tumor cell lines. Citation Format: Ilona Arnkil, Can Hekim, Elli Narvi, Emmi-Leena Ihantola, Endrit Elbasani, Anil Thotakura. The dual immunestimulatory and immunesuppressive role of MRC5 fibroblast cells in 3D coculture with T cells and tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4223.

Abstract 512: Cell-cycle inhibition may influence differences in 2D vs 3D tumor sensitivity profiles

Abstract Background: Traditional cell culture methods involve growing cells on flat surfaces, which do not fully replicate the human physiological conditions. In contrast, 3D cell cultures accurately reproduce aspects of the in-vitro malignant and non-malignant cell behavior. However, differences in tumor sensitivity platforms between 2D vs. 3D models might be relevant for some, but not for other drugs. Methods: The colorectal cancer cell line HCT-116 was cultured and plated in flat bottom well plates for the 2D condition and in Ultra Low Attachment round-bottom plates for the 3D condition. After plating, cells were treated with chemotherapies and targeted therapies for four days; cellular sensitivity was assessed through a viability assay. Four-parameter logistic (4PL) dose response curves were generated and IC50s were quantified to understand the differences in responses between 2D and 3D cellular environments. Results: HCT-116 exhibited similar responses to platinum drugs, taxanes, and several other tested drugs in both platforms, with the exception of gemcitabine (2D IC50 0.04 mcM; 3D IC50 12.8 mcM), lapatinib (2D IC50 23; 3D IC50 418), tucatinib (2D IC50 23.3; 3D IC50 79.3), and palbociclib (2D IC50 7.0; 3D IC50 24.0). Interestingly, these drugs share a common mechanism of action, which is the inhibition of cell cycle progression from G1 to S phase. Hence, we hypothesize that this shared mechanism may account for the observed differences in drug efficacy between the two environments. Conclusions: Similar dose responses have been observed with various drugs on both platforms, yet drugs that inhibit cell cycle progression from G1 to S phase, revealed higher sensitivity in 2D and more resistance in the 3D platform. Confirmatory cell-cycle results analyses are in progress. Further research is needed to understand if this applies to patient samples. Citation Format: Jahanvi Kumar, Chiara Maestri, Rajeshwar Nitiyanandan, Ivan Trus, Ricardo J. Parker, Brigitte Apfel, Christian Apfel. Cell-cycle inhibition may influence differences in 2D vs 3D tumor sensitivity profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 512.

Abstract 332: Improved high-throughput genomic analysis of urine samples using pixelated-ultrasound to enable personalized genomic profiling and therapeutic developments in cancer

Abstract Here we address some of the existing challenges in liquid biopsy workflows which can potentially benefit urine analysis from bladder cancer patients because approximately 1/4th of all human cancers are urological tumors. Globally, bladder cancer is the 10th most prevalent, and the 9th cause of mortality due to malignancy. The challenge addressed here is an improvement to sample preparation for sequencing. Among genders and disease states, urine samples vary in terms of cell count and hence, in DNA concentration. Therefore, it is critical to obtain consistent DNA fragments from variable samples across large cohorts of patients so that the sequencing leads to accurate identification of biomarkers of disease risk, progression, and treatment-response. In this direction, we present the advantages of a novel pixelated ultrasound technology which significantly improves sample-preparation in a very fast, economical, and high-throughput manner. Here, DNA is extracted from human urine samples and subjected to sonication to generate fragment sizes. Extracted DNA was loaded onto a regular 96-well round-bottom plate, which is sealed and placed inside the system where 12 columns of the 96-well plate are individually programmed and run simultaneously, hence enabling quick optimization. The system does not require time- and labor-intensive processing of the samples before or after the sonication which is required for other similar technology. Overall, the DNA samples were processed with significant improvement over other similar technology in terms of consistency, speed, and economy. Subsequently, the fragment sizes were analyzed. Analysis of results, as presented in the figures, show that the DNA fragment had the average size as predicted, uniform shearing, and very consistent across the 96-well plate containing different samples with no cross-contamination. The time, labour, expense, and risk turned out to be very low compared to other similar technology. The sizes were appropriate for library preparation and sequencing. The technology works consistently for both low and high cell numbers which is a critical factor for heterogenous samples from patients. In conclusion, the study presents improvements to library preparation for sequencing which fit into the larger workflow of non-invasive liquid biopsy using urine samples. The ability to obtain consistent DNA fragments in a quick and economical manner will greatly enable unique fingerprinting of an individual's cancer or cancer risk across large cohorts, with the potential for integration in automation workflows. Citation Format: Martha Evans-Holm, Nicolai Lumbres, Rwik Sen, Patricia Montilla-Perez, Marc A. Paradise, Peter Lentz. Improved high-throughput genomic analysis of urine samples using pixelated-ultrasound to enable personalized genomic profiling and therapeutic developments in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 332.

Technical report: liquid overlay technique allows the generation of homogeneous osteosarcoma, glioblastoma, lung and prostate adenocarcinoma spheroids that can be used for drug cytotoxicity measurements.

Introduction: The mechanisms involved in cancer initiation, progression, drug resistance, and disease recurrence are traditionally investigated through in vitro adherent monolayer (2D) cell models. However, solid malignant tumor growth is characterized by progression in three dimensions (3D), and an increasing amount of evidence suggests that 3D culture models, such as spheroids, are suitable for mimicking cancer development. The aim of this report was to reaffirm the relevance of simpler 3D culture methods to produce highly reproducible spheroids, especially in the context of drug cytotoxicity measurements. Methods: Human A549 lung adenocarcinoma, LnCaP prostate adenocarcinoma, MNNG/HOS osteosarcoma and U251 glioblastoma cell lines were grown into spheroids for 20days using either Liquid Overlay Technique (LOT) or Hanging Drop (HD) in various culture plates. Their morphology was examined by microscopy. Sensitivity to doxorubicin was compared between MNNG/HOS cells grown in 2D and 3D. Results: For all cell lines studied, the morphology of spheroids generated in round-bottom multiwell plates was more repeatable than that of those generated in flat-bottom multiwell plates. HD had no significant advantage over LOT when the spheroids were cultured in round-bottom plates. Finally, the IC50 of doxorubicin on MNNG/HOS cultured in 3D was 18.8 times higher than in 2D cultures (3D IC50 = 15.07 ± 0.3µM; 2D IC50 = 0.8 ± 0.4µM; *p < 0.05). Discussion: In conclusion, we propose that the LOT method, despite and because of its simplicity, is a relevant 3D model for drug response measurements that could be scaled up for high throughput screening.

Automated high-throughput, high-content 3D imaging of intact pancreatic islets.

Diabetes poses a global health crisis affecting individuals across age groups and backgrounds, with a prevalence estimate of 700 million people worldwide by 2045. Current therapeutic strategies primarily rely on insulin therapy or hypoglycemic agents, which fail to address the root cause of the disease - the loss of pancreatic insulin-producing beta-cells. Therefore, bioassays that recapitulate intact islets are needed to enable drug discovery for beta-cell replenishment, protection from beta-cell loss, and islet-cell interactions. Standard cancer insulinoma beta-cell lines MIN6 and INS-1 have been used to interrogate beta-cell metabolic pathways and function but are not suitable for studying proliferative effects. Screening using primary human/rodent intact islets offers a higher level of physiological relevance to enhance diabetes drug discovery and development. However, the 3-dimensionality of intact islets have presented challenges in developing robust, high-throughput assays to detect beta-cell proliferative effects. Established methods rely on either dissociated islet cells plated in 2D monolayer cultures for imaging or reconstituted pseudo-islets formed in round bottom plates to achieve homogeneity. These approaches have significant limitations due to the islet cell dispersion process. To address these limitations, we have developed a robust, intact ex vivo pancreatic islet bioassay in 384-well format that is capable of detecting diabetes-relevant endpoints including beta-cell proliferation, chemoprotection, and islet spatial morphometrics.

Influence of microbial polyamines on the IL-10 production by peripheral blood leukocytes of healthy donors

As is known, bacterial polyamines, which include cadaverine and putrescine, are capable of influencing the activity of immunocompetent cells in many ways. In particular, this situation is observed in long-term inflammatory diseases, especially with intensive reproduction of microorganisms capable of producing polyamines. It is of interest to study the production of one of the main anti-inflammatory cytokines, IL-10, under the influence of bacterial polyamines. For research, a population of mononuclear leukocytes was isolated from the peripheral blood of healthy donors by gradient centrifugation. The cell suspension was placed in a round-bottomed plates with preliminarily added polyamines at concentrations of 5, 25, 50, 75, and 100 mmol/L. Wells not containing polyamines were used as a control. After incubation for 72 h at 37 °C and 5% CO2, the supernatants were harvested and used to determine the concentration of IL-10. We used a kit for determining the concentration of IL-10 using the enzyme immunoassay method (Russia). Statistical analysis was performed using the Statistica 6.0 software package. In the case of a distribution close to normal, Student’s t-test was used; in the rest, the Mann–Whitney test was used to assess the significance of differences. Studies have shown that leukocytes in the presence of concanavalin A produce IL-10 at a concentration of 17.13±6.08 pg/mL. It has been established that under the influence of polyamines of bacterial origin, the production of IL-10 is enhanced only if putrescine and cadaverine are at concentrations of 50 mmol/L and higher. At low concentrations of polyamines, no significant increase in IL-10 production was detected. Since IL-10 is an anti- inflammatory cytokine, for which the analgesic effect is also known, it should be expected that with an increase in its concentration in the focus of invasion of opportunistic bacteria, the inflammatory process will develop latently, when the symptoms are mild. In general, it can be expected that polyamine-producing bacteria will contribute to the maintenance of few symptomatic inflammation.

Abstract 5362: AI-enabled novel workflow to evaluate T-cell activity in vitro using 3D spheroid models

Abstract T-cell therapies are designed to help our immune system eliminate cancer cells. Those include CAR T-cells (Chimeric Antigen Receptor engineered T-cells), tumor infiltrating lymphocytes (TIL), and other genetically modified T-cells. In recent years, the field of cell therapy has started to expand, including the launch of the first CAR T-cell therapies to treat blood cancer in 2017, which was a critical milestone in this field. Despite its boom, the discovery of novel immunotherapies that specifically enhance T-cell response against cancer cells remains a challenging task, limited by the absence of robust in vitro models to evaluate these immunotherapies throughout their development. The use of CAR T-cells on solid tumors has been lagging due to challenges that include tumor heterogeneity, immunosuppressive microenvironments, and the lack of unique tumor antigens that can be recognized by the CAR-T cells. As such, the ability to screen for CAR T-cells (e.g. with CRISPR) that effectively target and kill tumors is an area of active research. Here we describe a method for assessment of T-cell effect and penetration into the multi-cellular 3D tumor spheroids as a proof-of-concept model for in vitro CAR T assays. Spheroids were formed from Hela cell lines in 96-well round bottom plates. We activated the human peripheral blood mononuclear (PBMC) cells with PMA/ionomycin for 6 hours, then treated the spheroids with stimulated PBMC for 72 hours. Samples were imaged with a confocal imager every 2 hours for a 72 hour period. High-content imaging and analysis allowed us to observe and measure phenotypic changes in cancer spheroids and the process of T-cell penetration over a period of 72 hours. To optimize the workflow, we developed an image analysis approach that uses a deep learning-based segmentation model and a machine learning-based classification model to quantify the T-cell induced phenotypic changes in the spheroids using brightfield images. Our results show clear distinct phenotypic changes among different treatment groups and the feasibility of using AI-based analysis workflows to accurately predict the efficacy of T-cells in an in vitro assay. Moreover, the information associated with the penetration of T-cell into the 3D spheroids was explored by calculating the coordinates of the penetrated T-cells and T-cell movements from the nearest edge of the spheroids where the T-cell penetration speed was estimated. Our results show that the stimulated T-cells have much greater penetration speed compared to non-stimulated T-cells. Image analysis also allowed us to measure and quantitate the deterioration of cancer spheroids and changes in their morphology. Overall, our results show that the 3D spheroid models and the high-content analysis workflow may potentially be used as a metric to evaluate the efficacy of cell therapy in vitro. Citation Format: Zhisong Tong, Oksana Sirenko, Misha Bashkurov, Angeline Lim. AI-enabled novel workflow to evaluate T-cell activity in vitro using 3D spheroid models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5362.

Self-organized insulin-producing β-cells differentiated from human omentum-derived stem cells and their in vivo therapeutic potential

BackgroundHuman omentum-derived mesenchymal stem cells (hO-MSCs) possess great potential to differentiate into multiple lineages and have self-renewal capacity, allowing them to be utilized as patient-specific cell-based therapeutics. Although the use of various stem cell-derived β-cells has been proposed as a novel approach for treating diabetes mellitus, developing an efficient method to establish highly functional β-cells remains challenging.MethodsWe aimed to develop a novel cell culture platform that utilizes a fibroblast growth factor 2 (FGF2)-immobilized matrix to regulate the adhesion and differentiation of hO-MSCs into insulin-producing β-cells via cell–matrix/cell–cell interactions. In our study, we evaluated the in vitro differentiation potential of hO-MSCs cultured on an FGF2-immobilized matrix and a round-bottom plate (RBP). Further, the in vivo therapeutic efficacy of the β-cells transplanted into kidney capsules was evaluated using animal models with streptozotocin (STZ)-induced diabetes.ResultsOur findings demonstrated that cells cultured on an FGF2-immobilized matrix could self-organize into insulin-producing β-cell progenitors, as evident from the upregulation of pancreatic β-cell-specific markers (PDX-1, Insulin, and Glut-2). Moreover, we observed significant upregulation of heparan sulfate proteoglycan, gap junction proteins (Cx36 and Cx43), and cell adhesion molecules (E-cadherin and Ncam1) in cells cultured on the FGF2-immobilized matrix. In addition, in vivo transplantation of differentiated β-cells into animal models of STZ-induced diabetes revealed their survival and engraftment as well as glucose-sensitive production of insulin within the host microenvironment, at over 4 weeks after transplantation.ConclusionsOur findings suggest that the FGF2-immobilized matrix can support initial cell adhesion, maturation, and glucose-stimulated insulin secretion within the host microenvironment. Such a cell culture platform can offer novel strategies to obtain functional pancreatic β-cells from patient-specific cell sources, ultimately enabling better treatment for diabetes mellitus.Graphical

Antihelminthic Activity of Lophira Lanceolata on Heligmosomoides polygyrus Using an Automated High-Throughput Method.

There are about 13 parasitic infections that are responsible for significant morbidity and mortality but have not received the attention they deserve; thus, they are now known as "neglected tropical diseases" (NTDs). This study was aimed at evaluating the antihelminthic activities of Lophira lanceolata using an automated high-throughput method. The antihelminthic activity effect of the extracts against H. polygyrus was determined using an automated high-throughput method. For the egg-hatching test, 100 μL of embryonated egg suspension (60 eggs) was added to 100 μL of various concentrations of extracts, levamisole, and 1.5% DMSO in a 96-well round-bottom microtitre plate. After mixing, the 96-well microplate was placed in WMicroTracker and incubated for 24 h at 25°C; the movements were recorded every 30 minutes. The same procedure was used for the larval motility assays, where 100 μL of L1 or L2 larvae (50 larvae) were put in contact with 100 μL of various concentrations of extracts. The ovicidal activity (hatching) had an IC50 of 1.4 mg/mL for the ethanol extract. The aqueous and ethanol extracts of L. lanceolata showed larvicidal activity on the L1 larvae with IC50 of 1.85 mg/mL and 2.4 mg/mL, respectively, as well as on the L2 larvae with IC50 values of 1.08 mg/mL and 1.02 mg/mL for the aqueous and ethanol extracts, respectively. These results showed that the aqueous extract exhibited a stronger inhibitory power on the hatching rate of parasites than ethanol extracts, while the contrary effect was observed for the larval motility assays. This study provides scientific data on the use of L. lanceolata by the local population for the treatment of helminthiases. However, in vivo and toxicity tests are necessary to assess its activity and safety.

Analysis of the Sound Field Characteristics of a Muffler at Different Flow Conditions

To reduce the noise in a pump piping system and increase the usage time of equipment, a new type of porous muffler is proposed in this paper. A water guide cone is incorporated into the muffler structure, which may help to redirect the fluid media in the piping system. The porous structure is adapted from a muffler shell, water cone wall and round bottom plate. According to this structure, the muffler sample is made, and a pump pipeline system test bench is set up. The outlet noise of the pump pipeline system is measured after installing the muffler. At the same time, the muffler is numerically simulated by combining computational fluid mechanics and Lighthill acoustic theory. The characteristics of the flow field and external sound field under three different flow conditions of 200 m3/h, 400 m3/h and 600 m3/h are assessed. The numerical simulation results show the same dominant frequency and trend as the experimental results. The rationality of the numerical simulation is verified. Research shows that: the level of sound pressure at the muffler's outlet is lower than at the inlet, causing muffling, and the characteristics of a quadrupole sound source appear at the outlet. The proposed muffler has a certain effect in reducing noise in the pump pipeline system.

Optimisation of Conditions for the Formation of Spheroids of Head and Neck Squamous Cell Carcinoma Cell Lines for Use as Animal Alternatives.

The use of in vitro 3-D cell culture models in cancer research has yielded substantial gains in knowledge on various aspects of tumour biology. Such cell culture models could be useful in the study of head and neck squamous cell carcinoma (HNSCC), where mimicking intratumoral and intertumoral heterogeneity is especially challenging. Our research aims to establish 3-D spheroid models for HNSCC that reproduce in vitro the connections between tumour cells and the surrounding microenvironment. The aims of this study were to determine the optimal conditions for the culture and use of spheroids from HNSCC cell lines and optimal timepoint for using the spheroids obtained, to evaluate the effects of coculture with tumour-specific fibroblasts on spheroid formation, and to investigate spheroid responses to cisplatin treatment. Four HNSCC cell lines (UMSCC-11A, UMSCC-11B, UMSCC-22B and UD-SCC-01) were seeded in flat or round bottom well ultra-low attachment spheroid plates, and spheroid formation was evaluated. The HNSCC cell lines were then cocultured with stromal cells of the tumour microenvironment, producing an accelerated formation of dense spheroids. The viability of cells within the spheroids was assessed during cell culture by using a fluorescent dye. Our results suggest that: three out of the four cell lines tested could form usable spheroids with acceptable viability; the addition of stromal cells did not improve the number of viable cells; and the use of round bottom well plates supported the formation of a single spheroid, whereas flat bottom well plates led to the formation of multiple spheroids of different sizes.

Increase in Anticancer Drug-Induced Toxicity by Fisetin in Lung Adenocarcinoma A549 Spheroid Cells Mediated by the Reduction of Claudin-2 Expression.

Claudin-2 (CLDN2), a component of tight junction, is involved in the reduction of anticancer drug-induced toxicity in spheroids of A549 cells derived from human lung adenocarcinoma. Fisetin, a dietary flavonoid, inhibits cancer cell growth, but its effect on chemosensitivity in spheroids is unknown. Here, we found that fisetin (20 μM) decreases the protein level of CLDN2 to 22.3%. Therefore, the expression mechanisms were investigated by real-time polymerase chain reaction and Western blotting. Spheroids were formed in round-bottom plates, and anticancer drug-induced toxicity was measured by ATP content. Fisetin decreased the phosphorylated-Akt level, and CLDN2 expression was decreased by a phosphatidylinositol 3-kinase (PI3K) inhibitor, suggesting the inhibition of PI3K/Akt signal is involved in the reduction of CLDN2 expression. Hypoxia level, one of the hallmarks of tumor microenvironment, was reduced by fisetin. Although fisetin did not change hypoxia inducible factor-1α level, it decreased the protein level of nuclear factor erythroid 2-related factor 2, a stress response factor, by 25.4% in the spheroids. The toxicity of doxorubicin (20 μM) was enhanced by fisetin from 62.8% to 40.9%, which was rescued by CLDN2 overexpression (51.7%). These results suggest that fisetin can enhance anticancer drug toxicity in A549 spheroids mediated by the reduction of CLDN2 expression.

Improvement of antimicrobial susceptibility testing in biofilm-growingcoagulase-negative Staphylococcus hominis

Coagulase-negative Staphylococcus hominis causes bloodstream infections and often can form biofilms on medical devices. This study aimed to improve the current methodology for antimicrobial susceptibility testing (AST) in biofilm-growing S. hominis isolates. Biofilm production of S. hominis was assessed using the crystal violet staining method in trypticase soy broth supplemented with 1% glucose (TSBglu1%), Mueller-Hinton broth (MHB), or MHBglu1% using flat-bottom plates or the Calgary device. Susceptibility to antibiotics was assessed using the broth microdilution method (MHB and TSBglu1%) in planktonic cells (round-bottom plates) and biofilm cells (flat-bottom plates and the Calgary device). Biofilm determination using TSBglu1% yielded better performance over MHB, and flat-bottom plates without agitation were preferred over the Calgary device. Higher fold dilution values between the minimum biofilm eradication concentration (MBEC) and the minimum inhibitory concentration (MIC) were obtained in MHB for almost all antibiotics, except for linezolid. TSBglu1% and flat-bottom polystyrene plates were preferred over MHB and the Calgary device for biofilm determination. AST in biofilm-growing S. hominis showed better performance using TSBglu1% compared to MHB. Therefore, when comparing MBEC and MIC values, AST in planktonic cells could also be performed using TSBglu1% instead of MHB.

Matrix-free three-dimensional culture of bovine secondary follicles to antral stage: Impact of media formulation and epidermal growth factor (EGF)

Disrupted/disordered ovarian steroidogenesis is associated with several fertility disorders such as polycystic ovary syndrome in humans and cystic ovarian disease in cattle. Methods to interrogate theca cell processes as part of follicular development are necessary to further research into treatments for these types of disorders. Multilayer follicles of dairy-breed cows were placed into culture in a novel matrix-free 3D system using round bottom low-attachment plates. Follicles were first cultured in the presence of two types of media previously used for in vitro follicle maturation (basal α-MEM and basal T-199). After the optimal media was identified, impact of supplementation of epidermal growth factor (EGF) on growth and survival of bovine secondary follicles to antral stage was evaluated. No differences were observed in growth and survival of follicles cultured in basal α-MEM media or basal T-199 media, although T-199 media's high phenol red content made assessment of follicles difficult. Further studies were then performed with α-MEM media. Three cohorts of follicles were observed based on time to antrum formation: ≤ 5 days (fast), 6–19 days (slow), or survived but did not form an antrum by 21 days (no). Supplementation of EGF to the basal α-MEM media dramatically improved follicle survival rates in culture (defined as follicles that either formed an antrum or did not form an antrum but did not die during 21 day culture period) from 29% to 95.7% (Chi-square p < 0.0001). However, in follicles that survived to form an antrum there were no differences in proportion of fast, slow and no antrum follicles after addition of EGF (Chi-square p > 0.7). Fast antrum follicles treated with EGF plateaued in size earlier in culture compared to controls (p = 0.013). Slow and no antrum follicles were larger in diameter during EGF culture than controls (p's < 0.0001). Many follicles cultured in this matrix-free system that formed an antrum approached 1.5–2 mm in size, an improvement from previous single follicle culture methods used for bovine pre-antral follicles in vitro. In addition, follicles displayed functional steroidogenesis in vitro producing measureable levels of estradiol and androstenedione. This matrix-free 3D culture system provides an excellent in vitro model to explore processes associated with folliculogenesis in cattle.

Functional Immune Tolerance Induced By Sequential Hematopoietic Stem Cell-Solid Organ Transplantation

Solid organ transplantation (SOT) treats over 1500 children/year in the US. While short-term outcomes have improved, long-term outcomes still remain poor. With the rare exception of monozygotic twins as donors, recipients require lifelong immunosuppression with its accompanying toxicities, the risk of nonadherence, and chronic rejection. Further, up to half of recipients require a second transplant.We have pioneered translational approaches that abrogate rejection and the need for post-transplant immunosuppression. Previous attempts in adult patients utilizing allogeneic hematopoietic stem cell transplantation (HSCT) to eliminate the need for post-SOT immunosuppression in non-histocompatible kidney transplant (KT) recipients have failed(Lowsky R, BMT 2019). The establishment of mixed lymphoid chimerism has permitted a reduction in the number of immunosuppressive drugs needed, but not their discontinuation, whereas complete donor engraftment after HSCT has resulted in fatal graft-versus-host disease in some patients (Leventhal JR, Eur J Pediatr 2000). The use of HSCT to eliminate the need for immunosuppression following pediatric KT has not been evaluated. We have pioneered sequential haploidentical HSCT followed by KT in three patients with Schimke immuno-osseous dysplasia (SIOD) to abrogate kidney rejection and the need for post-transplant immunosuppression.SIOD is a monogenic disorder with progressive nephropathy correctable by KT and a T-cell immunodeficiency curable by HSCT. All three patients received αβT-cell/CD19 B-cell depleted HSCT (αβhaplo-HSCT) from a parent prior to a KT from the same parental donor. The pre-HSCT reduced intensity preparative regimen consisted of fludarabine (a starting dose of 1 mg/kg x 4 days, which was adjusted based on AUC), total body irradiation (TBI) 200 cGy, cyclophosphamide 1200 mg/m 2, anti-thymocyte globulin (ATG) thymoglobulin® 7.5 mg/kg, and rituximab 200 mg/m 2. Immunosuppressive drugs were not prophylactically administered after HSCT. After confirmation of full donor lymphoid chimerism post-HSCT, the patients received a living donor KT from their parental HSCT donor. Post-KT immunosuppression included intraoperative methylprednisolone and post-operative low-dose oral prednisone (0.5 mg/kg/day with taper) and tacrolimus (target serum level of 3-5 ng/ml) to reduce potential reperfusion inflammation. All immunosuppressive drugs were tapered off by Day +30 post-KT. To demonstrate functional tolerance after KT, Mixed Lymphocyte Cultures (MLC) were performed between peripheral blood mononuclear cells (PBMC) and patient/parent-derived irradiated EBV transformed lymphoblastoid cells lines (EBV-LCL) as stimulators (Figure 1). PBMC were isolated from SIOD patients (n=3) by Ficoll Hypaque separation and were labelled with Invitrogen™ CellTrace Violet Proliferation Kit (CTV). EBV-LCL were established from SIOD patients, parents and healthy donors. The assay was performed in round bottom microtiter plates with 100,000 labelled PBMC as responder cells (R) and 50,000 irradiated (30gy) EBV-LCL in RPMI medium with 10% fetal calf serum. Cells were harvested on Day 6, and T-cell proliferation determined by CTV expression of CD3+ T cells using a Novocyte Penteon flow cytometer (Agilent).Donor-derived T cells isolated from the peripheral blood of all three recipients > 1-year post-HSCT/KT, did not respond to the donor cells, while they did proliferate to the non-donor parental and control cells (Fig. 1). These results demonstrate that post-HSCT/KT the circulating donor-derived T cells are functionally tolerant to the transplanted kidney and, therefore, are unable to mediate graft rejection even in the absence of immune suppression. We have achieved two goals: first, ablating the recipient immune system before HSCT we safely established a new donor-derived immune system - that could also, if required, cure a patient's underlying disease- and, second, functional tolerance to the transplanted kidney.At 12 to 24 months after KT, all patients have full donor lymphoid and myeloid chimerism, normal renal function without immunosuppression, MLC demonstrated tolerance towards donor cells, and normal proliferative responses to non-donor parental cells. These findings suggest that the combination of αβhaplo-HSCT and SOT could be extended to other solid transplantation like liver and small intestine. [Display omitted] DisclosuresBertaina: Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; AdicetBio: Membership on an entity's Board of Directors or advisory committees. Shah: OrchardTherapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Dr. Shah currently serves on the medical advisory board for Orchard Therapeutics . Parkman: Jasper Biotech: Consultancy.

Abstract A55: Pancreatic stellate cells promote pancreatic cancer invasion and metastasis by secretion of soluble factors and through contact-mediated mechanisms

Abstract Pancreatic ductal adenocarcinoma (PDAC) is rarely detected early, instead presenting in patients as advanced metastatic disease once symptoms are evident. Given the central role of metastasis in pancreatic tumor progression and mortality, there is an urgent need for further investigation of how PDAC metastasizes and for novel treatment strategies targeting PDAC metastasis. A prominent histopathologic feature of PDAC is a desmoplastic, fibrotic stroma consisting of extracellular matrix components, immune cells, and activated fibroblasts termed “pancreatic stellate cells” (PSCs) that often compose a majority of cellular content in many PDAC tumors. Data from recent studies demonstrate multiple phenotypic PSC populations in the stroma (Ohlund et al., 2017); however, the influence of individual PSC populations on metastasis and disease progression is not fully understood. We hypothesized that PSCs promote pancreatic tumor metastasis through secretion of soluble factors and accompany these metastasizing tumor cells to distal sites using collective invasion mechanisms. Tumor spheroids were formed by centrifugation of HPAC cells (a human PDAC cell line) in low adhesion round bottom plates. Spheroids were embedded in both collagen- and Matrigel-based matrices and z-stacks were collected every 24hrs for 72hrs using a Lecia SP8 confocal microscope. Live cell imaging was performed in an incubation chamber for 72hrs using a Leica SP8 confocal microscope. Human pancreatic stellate cells were isolated from surgically resected tumor specimens at Emory University Hospital under IRB-approved protocols and grown to ~70% confluency before supernatants were collected. Utilizing innovative 3D invasion assays, we demonstrate the ability of human PSC conditioned media to initiate and promote invasion of HPAC pancreatic tumor spheroids. HPAC spheroids exposed to 10% PSC supernatants showed significantly decreased roundness in comparison to media alone. Further investigation of possible soluble factors mediating this interaction indicated a protein-based interaction, although alternative mechanisms by which metastasis may be initiated were also identified. Phenotypic analysis of invasion through live cell imaging led to observations of unique invasion mechanisms by which hyperactive cells at the leading edge seemingly guide invasion of large groups of cells. Additionally, we observed co-culture interactions between PSCs and HPAC cells. In comparison to HPAC cells alone, ratios of 10:1 PSCs to HPAC cells increased circularity (less invasive) while inverse ratios decreased circularity of spheroids (more invasive). This study supports the hypothesis that PSCs can promote collective invasion and migration of pancreatic tumor cells through both contact-dependent and -independent means. Further identification of mechanisms mediating this interaction may reveal novel therapeutic targets for the treatment of PDAC and present new strategies for targeting metastasis of this deadly disease. Citation Format: Michael B. Ware, Jerry Yue, Bassel El-Rayes, Shishir K. Maithel, Juan Sarmiento, Gregory B. Lesinski. Pancreatic stellate cells promote pancreatic cancer invasion and metastasis by secretion of soluble factors and through contact-mediated mechanisms [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A55.

The biofilm formation of freshly isolated and vaccine strains of Bordetella pertussis of different serotypes

Aim. To study the formation of biofilms by freshly isolated and vaccine strains of Bordetella pertussis of different serotypes.Materials and methods. The intensity of biofilm formation by B. pertussis strains in 96-well round-bottom polystyrene plates by using three sowing doses of microbial cells (1,25 IOU/ml, 2,5 IOU/ml and 5,0 IOU/ml) was estimated by staining with 0,1% gentian-violet solution. The results were interpreted after measuring the optical density (OP) of the colored solvent at a wavelength of 600 nm.Results. The highest intensity of biofilm formation was found in the newly isolated strain No. 211 and vaccine strain No. 475, both belonging to serotype 1.2.3. Cultures of these strains formed dense biofilms at all sowing doses of microbial cells. Certain differences in the intensity of biofilm formation were found between freshly isolated and vaccine strains of serotypes 1.2.0 and 1.0.3, especially when using a sowing dose of 5,0 IOU/ml. Freshly isolated strains at this dose formed dense biofilms, while two of the three vaccine strains formed moderate biofilms, and one strain was dense.Conclusion. The revealed differences between the strains of B. pertussis in the intensity of biofilm formation may be associated with the peculiarities of the expression of agglutinogens, as well as other surface structures of microbial cells involved in the adhesion process on the substrate.

Coculture with noncardiac cells promoted maturation of human stem cell-derived cardiomyocyte microtissues.

Cardiomyocytes derived from human pluripotent stem cells (hPSC-CM) provided a promising cell source for cell therapy, drug screening, and disease modeling. However, hPSC-CM are immature and phenotypically more similar to fetal rather than adult cardiomyocytes in vitro. We explored the impact of coculture of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSC) and endothelial cells (ECs) with human embryonic stem cells-derived cardiac progenitor cells (hESC-CPC) on the gene expression and electrophysiological properties of hESC-CPC in 3D culture (microtissue spheroid). In this regard, hESC-CPC were cultured either alone (CM microtissue) or in coculture with EC and hESC-MSC (CMEM microtissue) on agar-coated 96-well round-bottomed plates for 1 week. Lumen-like structures were formed in CMEM but not in CM microtissue. Cardiac progenitor markers (TBX5, GATA4) were downregulated and cardiac sarcomeric transcripts (MLC2v and β-MHC) were upregulated in CMEM compared with CM microtissue. Furthermore, beating frequencies, beating cycles, and field potential durations of CMEM resided in the range of adult cardiomyocytes rather than fetal like phenotypes observed in CM microtissue. These findings demonstrated that CPC spheroids in coculture with EC and hESC-MSC may undergo greater maturation toward an adult-like cardiomyocyte.

Comparative In vitro Activities of Several Oxazolidinones against Mycobacterium kansasii

Background: Oxazolidinones were found to be active in vitro against mycobacteria particularly M. tuberculosis soon after their discovery. Linezolid (LZD) has been effective in the treatment of patients with multidrug resistant tuberculosis, however, significant toxicity issues limit its use in tuberculosis and other mycobacterial diseases. Newer oxazolidinones, which may have reduced toxicity, are at various stages of development. The purpose of the present study was to evaluate the in vitro activities of MRX-1, sutezolid, and tedizolid compared to linezolid against clinical isolates of M. kansasii (MK). Methods & Materials: Linezolid and MRX-1 were provided by MicuRx Pharmaceuticals (Hayward, CA), tedizolid was provided by Trius Pharmaceuticals (San Diego, CA) and sutezolid was provided by NIAID (Bethesda, MD) Each of the drugs were dissolved in 100% dimethyl sulfoxide (DMSO) at a concentration of 5 mg/ml prior to freezing at -20 °C. MK isolates (N = 20) were grown in Middlebrook 7H9 broth with 10% ADC. The organisms were diluted in Middlebrook 7H9 broth to about 5 × 105 CFU/ml for use in the broth dilution assay. The actual inocula used for each isolate was determined by titration and plating on 7H10 agar plates which were incubated at 370 C for 2 weeks prior to counting. Polystyrene 96-well round-bottom microtiter plates were filled with 50 μl of broth. The compounds were prepared at 4 times the maximum concentration at which they were to be tested and added to the first well prior to being serially diluted 2-fold. Fifty microliters of the inocula was added to each well. The microtiter plates were sealed and incubated at 37 °C in ambient air for 7 days prior to reading. Each isolate was tested in duplicate. The MIC was defined as the lowest concentration of antimicrobial agent yielding no visible turbidity. Results: Sutezolid was the most active agent with MIC50 and MIC90 respectively of 0.125 μg/ml and 0.25 μg/ml compared to 0.5 μg/ml and 0.5 μg/ml for tedizolid, 1 μg/ml and 1 μg/ml for MRX-1 and 1 μg/ml and 2 μg/ml for linezolid. Conclusion: These newer oxazolidinones should be studied in a murine model of pulmonary MK infection to determine if subsequent clinical evaluation is warranted.

Characterization of Gastrospheres Using 3D Coculture System.

To understand the molecular mechanisms involved in gastric disorders and regeneration, we need an in vitro tridimensional (3D) culture model, which can mimic the in vivo gastric microenvironment. A 3D coculture system named gastrosphere is proposed herein, composed of primary human gastric epithelial and stromal cells. The primary cultures were obtained from endoscopic gastric biopsies, and after mechanical and enzymatic dispersion, epithelial (HGE3) and stromal (HGS12) cells were expanded. After extensive immunocytochemical characterization, cells were seeded onto 96-well round bottom plates previously covered with 1% agarose. Cells were cultured in KM-F12 culture medium with 10% fetal bovine serum (FBS), antibiotics, and antimycotics, in humidified air at 37°C and atmosphere containing 5% CO2 for 72h or until spheres formation. Then gastrospheres were carefully transferred to a rotary cell culture system (RCCS-4), and maintained for 07, 14, 21, and 28days. Gastrospheres were morphologically characterized by immunocytochemistry [cytokeratins (CK), vimentin, α-smooth muscle actin (α-SMA), laminin (LN), fibronectin (FN), and type IV collagen (CIV), proliferating cell nuclear antigen (PCNA)], and electron microscopy. In gastrospheres, the cytokeratin-positive epithelial cells were found in the outer layer, while vimentin-positive stromal cells were localized in the center of the gastrospheres. PCNA+ cells were mainly seen at the peripheral and in the intermediary region while nestin+ cells were also depicted in the latter zone. Scanning electron microscopy revealed groups of cohesive gastric cells at the periphery, while transmission electron microscopy demonstrated some differentiated mucous-like or zymogenic-like cells in the periphery and stromal structures located at the center of the 3D structures. Extracellular matrix was deposed between cells. Our data suggest that in vitro gastrospheres recapitulate the in vivo gastric microenvironment.